DOMAIN BEHAVIOR DURING THE FOLDING OF A THERMOSTABLE PHOSPHOGLYCERATEKINASE

Bacillus stearothermophilus phosphoglycerate kinase (bsPGK) is a monom eric enzyme of 394 residues comprising two globular domains (N and C), covalently linked by an interdomain ex-helix (residues 170-185). The molecule folds to the native state in three stages. In the first, each domain rapidly and independently collapses to form an intermediate in which the N-domain is stabilized by 5.1 kcal mol(-1) and the C-domain by 3.3 kcal mol(-1) over their respective unfolded conformations. The N-domain then converts to a folded state at a rate of 1.2 s(-1) (Delt a G(I-F) = 3.8 kcal mol(-1)), followed by the C-domain at 0.032 s(-1) (Delta G(I-F) = 12.1 kcal mol(-1)). It is this last step that limits t he rate of acquisition of enzyme activity. In the dynamics of unfoldin g in water, the N-domain converts to the intermediate state at a rate of 8 x 10(-4) s(-1), some 10(7) times faster than the C-domain. Conseq uently, the most populated intermediate in the folding reaction has a native-like N-domain, while that in the unfolding direction has a nati ve-like C-domain. In a conventional sense, therefore, the folding/unfo lding kinetics of bsPGK can be described as random order. Consistent w ith these observations, cutting the molecule in the interdomain helix produces two, independently stable units comprising residues 1-175 and 180-394. A detailed comparison of their folding behavior with that of the whole molecule reveals that true interdomain contacts are relativ ely weak, contributing similar to 1.4 kcal mol-l to the stability of t he active enzyme. The only interactions which folding pathways are tho se within domain cores. Contacts formed either between domains or with the interdomain helix are made only in the folded ground state, but d o not constitute a separate step in the folding mechanism Intriguingly , the most pronounced effect of interdomain contacts on the kinetics o f folding is inhibitory; the presence of the C-domain appearing to red uce the effective rate of acquisition of native structure within the N -domain.